Protective effects of dietary Kefir against aflatoxin B1‐induced hepatotoxicity in Nile tilapia fish, Oreochromis niloticus

Abstract The effect of dietary Kefir supplementation on the biometric, biochemical, and histological parameters of Nile tilapia (Oreochromis niloticus) exposed to aflatoxin B1 (AFB1, 200 µg/kg diet) contamination was studied. The yeasts were dominant in Kefir followed by lactic and acetic acid bacteria. The Kefir showed relatively interesting antioxidant potential in the DPPH• (IC50 = 0.9 ± 0.02 mg/ml) and ABTS•+ (IC50 = 2.2 ± 0.03 mg/ml) scavenging activities, Fe3+‐reducing power (EC0.5 = 1.2 ± 0.01 mg/ml), and β‐carotene bleaching assay (IC50 = 3.3 ± 0.02 mg/ml). Three hundred and sixty Nile tilapia weighing 23 ± 5 g were divided into four groups (30 fish/group with 3 replicates), and fed with diets containing Kefir (D2), AFB1 (D3), and Kefir+AFB1 (D4) for 4 weeks, whereas D1 was kept as control group where fish were fed with basal diet. The Kefir supplementation in D4 group significantly increased (p < .05) the percent weight gain as compared to D3 group. Moreover, Kefir improved the antioxidant enzymes in the liver, such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities, that significantly increased (p < .05) by 2‐, 3‐, and 1.5‐folds, respectively, as compared to D3 group. The Kefir treatment significantly decreased (p < .05) the liver malonaldehyde content by ~50% as compared to D3 group. Histopathological analysis revealed the hepatoprotective effects of Kefir by showing normal liver histological architecture in D4 group, as compared to degenerative changes observed in D3 group. These results suggest that Kefir could be considered as a potential probiotic in Nile tilapia feed to mitigate the AFB1 harmful effects.

In Tunisia, as a coastal country, fish production is increasing year by year and it reached 1327 tons in 2020. Farmed fish occupied about two thirds of this production, the rest was made up of mollusks and crustaceans. However, fish production has suffered from mycotoxins in the feed during the storage (Ayofemi et al., 2020). The aflatoxins, which were metabolites produced by Aspergillus species, such as A. flavus and A. parasiticus, represented one of the most important mycotoxins types that attack fed fish (Singh & Mehta, 2020).
In recent decades, food contamination with naturally occurring toxic compounds has become a major concern worldwide (Noroozi et al., 2020). Thus, the management of mycotoxin contamination in the fish feed industry was tricky (Yitbarek & Tamir, 2013). In fact, many chemical and physical procedures were used to control mycotoxins in fish feed. However, excessive use of mycotoxin killers could destroy nutrients and lead to a loss of organoleptic quality. The biocontrol of mycotoxins using the catabolic potential of lactic acid bacteria (LAB) and yeasts could be an encouraging way (Mohammadi et al., 2021). The Kefir, as a complex symbiotic association of yeasts and LAB (Ben Taheur et al., 2017), could be a promising avenue for the management of aflatoxin B1 (AFB1) in tilapia fish.
To the best of our knowledge, the effect of Kefir as a dietary supplement in mitigating AFB1 toxicity has not been studied. Thus, the present study aimed to explore some in vitro and in vivo properties of Kefir. The in vitro antioxidant potential was evaluated through various complementary methods, such as Fe 3+ -reducing power, βcarotene bleaching assay, radical (DPPH• and ABTS•+)-scavenging activities, and DNA nicking assay. The protective effects of Kefir administration in tilapia fish on antioxidant enzymes, lipid peroxidation, and hepatic histoarchitecture in AFB1-induced toxicity was studied.

| Grain activation and Kefir production
The Kefir grains used in this study were a traditional culture, and the original grain was from the Laboratory of Analysis, Treatment and

Valorization of Environmental Pollutants and Products (Faculty of
Pharmacy of Monastir, University of Monastir, Monastir, Tunisia). Kefir grains were activated by adding 20 g of grains to 200 ml of commercial ultra-high-temperature (UHT) cows' milk, followed by incubation at 25°C. After 24 h, the grains were sieved out and rinsed with sterile distilled water to remove the clotted milk. Then, Kefir grains were placed in 200 ml of fresh UHT milk and incubated under the same conditions. This step was repeated every day for 2 weeks until the grains were used as starter culture in the following experiments (Ben Taheur et al., 2017).
The Kefir fermented milk was lyophilized and stored at −20°C until use.

| Microbiological analyses
Enumeration and isolation of microorganisms from fermented milk were performed (Ben Taheur et al., 2017). After incubation period of activated grains at 25°C for 24 h in milk, a volume of 10 ml of Kefir was homogenized with 90 ml of physiological sterile water.
Serial decimal dilutions were prepared, and 0.1 ml was inoculated in triplicate by surface spreading on specific solid media. LAB were isolated on De Man-Rogosa-Sharpe (MRS) agar (Merck) and incubated at 37°C under anaerobic conditions for 3 days. Yeasts were isolated on potato-dextrose-agar (PDA) (Merck) at 30°C for 3 days.

| DPPH• radical-scavenging activity
The DPPH• (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity of Kefir was determined using the method described by Kirby and Schmidt (1997). Briefly, 500 µl of Kefir sample at various concentrations (1-5 mg/ml) was mixed with 375 µl ethanol and 125 µl of DPPH• solution (0.02% (w/v) prepared in ethanol). For each concentration, a blank without DPPH• and a control without sample were prepared. After incubation for 60 min in the dark, the absorbance was measured at 517 nm using UV/VIS T-80 spectrophotometer (PG Instruments Limited). The IC 50 value, defined as Kefir concentration needed to scavenge 50% of DPPH•, was determined. BHA (Butylated hydroxyanisole) was used as a positive standard, and the DPPH• reduction was calculated using the Equation (1).
where A C , A b , and A S represent the absorbances of the control, blank, and sample reaction tubes, respectively.

| Ferric-reducing power
The method described by Yildirim et al. (2001) was used to determine the ferric-reducing power of Kefir. This assay consisted of estimating the capacity of antioxidant compounds in Kefir to reduce ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ). Kefir sample (500 µl) at different concentrations (1-5 mg/ml) was mixed with 1.25 ml 0.2 M sodium phosphate buffer (pH = 6.6) and 1.25 ml 1% (w/v) potassium ferrocyanide (K 3 Fe(CN) 6 ). The mixture was incubated for 30 min at 50°C. Afterward, 1.25 ml 10% (w/v) of trichloroacetic acid (TCA) was added and the mixture was centrifuged at 11,000 × g for (1) 10 min. Then, for each concentration sample, 1.25 ml of the resulting supernatant was mixed with 1.25 ml distilled water and 0.25 ml 0.1% (w/v) ferric chloride solution. Absorbance was measured at 700 nm using UV/VIS T-80 spectrophotometer (PG Instruments Limited). Blanks (without FeCl 3 ) were prepared as described previously and the BHA was used as a standard. The reducing power represented the absorbance measured at 700 nm, and the Kefir sample concentration providing 0.5 of the initial absorbance (EC 0.5 ) was calculated from the graph of absorbance at 700 nm against sample concentration.

| β-Carotene bleaching assay
The β-carotene bleaching activity was evaluated using the βcarotene-linoleic acid assay as described by Koleva et al. (2002). where A S0 : absorbance of the sample at t = 0, A S2 : absorbance of the sample at t = 2 h, A C0 : absorbance of control at t = 0, A C2 : Absorbance of control at t = 2 h.

| ABTS•+ radical-scavenging activity
The ABTS•+ (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) radical-scavenging activity of Kefir was determined according to the colorimetric method described by Re et al. (1999). ABTS•+ stock solution (7 µM) was mixed with 2.45 µM potassium persulfate solution and incubated in the dark at room temperature for 12-16 h. The absorbance of the ABTS•+ solution at 734 nm was adjusted to 0.700 (±0.02) using UV/VIS T-80 spectrophotometer (PG Instruments Limited) and equilibrated at 30°C. A volume of 3 ml of diluted ABTS•+ solution was added to 30 µl of Kefir sample at different concentrations (1-5 mg/ml) and the absorbance was measured at 734 nm after 6 min (A t ). The mixture without sample was used as reagent blank reading (A 0 ). The IC 50 value, defined as the Kefir sample concentration needed to scavenge 50% of ABTS•+, was determined.
The Trolox was used as a standard. The antioxidant activity of Kefir was expressed in terms of inhibition as given by Equation (3).
where A: the absorbance of ABTS•+ solution and A 0 : the absorbance of ABTS•+ solution with Kefir sample.

| Experimental diets
The diet (Table 1)  was added gradually with mixing, until the mixture was molded into small balls. After that, the mixture was divided into four diets. The basal diet D1 was considered as control diet. The D2, D3, and D4 diets were supplemented with Kefir, AFB1, and Kefir+AFB1, respectively, as mentioned in Table 1. The diets were mixed very well to ensure that Kefir and/or AFB1 were evenly distributed throughout the mixture. Then, the diets were ground to a fine powder using a laboratory hammer mill, sieved through a 0.25 mm sieve, and dried at room temperature for 24 h. Finally, the dried diets were stored at −20°C until use.

| Fish and experimental protocol
The experimental protocol was conducted at the fish-culture research station of the National Institute of Marine Sciences and Technologies (Bechima, Gabes, Tunisia). Three hundred and sixty male Nile tilapia (Oreochromis niloticus L.), weighing 23 ± 5 g, were randomly maintained in twelve 400 L plastic rearing tanks in batches of 30 fish/tank. The tanks were supplied with water from a geothermal source after undergoing cooling in a large storage tank located upstream of the experimentation system. Three replicate tanks/dietary treatment were used. At the beginning, fish were (2) β−carotene bleaching inhibition activity (%) acclimatized for 15 days prior to the experimental feeding trial, and they were fed a basal diet (D1) daily (Table 1) at a rate of 3% of the body weight/day (Azaza et al., 2008). Then, fish were divided into four groups (30 fish/treatment; three replicates/tank) representing four nutritional groups. The fish of one group remained on the basal diet and were considered as control animals. The other fish groups were switched to the treatment diet containing Kefir (D2), AFB1 (D3), and Kefir+AFB1 (D4). The fish were fed three times daily for 4 weeks. For monitoring growth, the mean body weight/tank was determined weekly by measuring the weight of all the fish in each tank. Water temperature and dissolved oxygen were measured daily using a WTW/Oxi 96 oxythermometer (WTW Measurement Systems Inc.). Levels of pH, nitrite, nitrate, and total ammonium were measured weekly. All measured parameters remained within the tolerance limits known for tilapia. At the end of the experimental period, 30 fish were randomly selected from each treatment (10 from each tank), weighed, and anesthetized with clove essential oil.
The livers were removed and weighed. For each fish, a slice of liver was fixed in 10% formaldehyde solution for histological analysis and the rest of the tissues were stored at −20°C for biochemical analyses.
where FW: final body weight and IW: initial body weight.
where FW: final body weight and IW: initial body weight.
where LW: liver weight and FW: final body weight.

| Biochemical measurements
All measurements were performed at 4°C to prevent enzyme or tissue degradation. Frozen liver tissues were thawed and homogenized in Tris-HCl buffer (50 mM Tris-HCl, 150 mM NaCl, pH = 7.4) in a 1:3 (w/v) ratio using a homogenizer. The homogenates were centrifuged for 25 min at 9,000 × g at 4°C, and the resulting supernatants were immediately frozen at −20°C until use. Each biochemical measurement was performed on 20 individual livers from each experimental group and each determination was performed in triplicate.

| Total protein content
The total protein content of homogenate samples was measured according to the method of Bradford (Bradford, 1976)   VIS T-80 spectrophotometer (PG Instruments Limited) at 595 nm.

| Catalase (CAT) activity
The CAT activity was measured based on the method adapted from (Aebi, 1984

| Glutathione peroxidase (GPX) activity
The GPX activity was measured in a coupled enzyme system where NADPH was consumed by glutathione reductase to convert the formed oxidized glutathione form (GSSG) to its reduced form (GSH) according to the method described by Flohé and Günzler (1984) with slight modifications. The decrease of absorbance was monitored at 340 nm using UV/VIS T-80 spectrophotometer (PG Instruments Limited). The GPX activity was expressed in nmol/ min mg −1 protein.

| Superoxide dismutase (SOD) activity
The SOD activity was determined according to the method described by Sun et al. (1988). The SOD amount capable to inhibit 50% of the reduction rate of nitroblue tetrazolium (NBT) in presence of superoxide anion radicals generated by riboflavin/methionine reaction was measured at 580 nm using UV/VIS T-80 spectrophotometer (PG Instruments Limited). The enzymatic activity was expressed as unit mg −1 protein.

| Lipid peroxidation quantification
The lipid peroxidation was estimated from the formation of thiobarbituric acid reactive substance (TBARS) using the method described by Yagi (1976). The TBARS, considered as malonedialdehyde (MDA)like peroxide products, were quantified spectrophotometrically by reference to MDA absorbance at 530 nm using UV/VIS T-80 spectrophotometer (PG Instruments Limited). The lipid peroxidation was expressed in nmol MDA equivalent mg −1 protein.

| Histological analysis
The livers from control and treated fish were dissected and immediately fixed with 10% formaldehyde solution. After dehydration in a graduated alcohol series, samples were embedded in paraffin. Thin sections of 3 μm were cut, mounted onto glass slides, and subjected to hematoxylin and eosin (H&E) staining for histopathological examination. Preparations were observed and micro-photographed at 40 × using a Leica Orthoplan microscope (Leica, Solms, Germany) to assess any liver alterations in treated fish as compared to controls.

| Statistical analyses
The data were analyzed using a one-way analysis of variance (ANOVA) to determine differences between experimental groups.
Significant differences were determined at the p < .05 level using Tukey test. The principal component analysis (PCA) was used to find correlations between the different biomarkers. Statistical analysis was performed using the software Statistica 6.1.478.0 (StatSoft Inc).

| Microbiological analyses
The total counts of viable microorganisms in Kefir are shown in

| Antioxidant potential
The antioxidant activities of Kefir were measured using four complementary tests: (i) DPPH• radical-scavenging activity; (ii)
However, these activities remained lower than the artificial antioxidant BHA. Interestingly, the obtained results were higher than those obtained with peanut-milk Kefir, where the IC 50 value for DPPH• radical-scavenging activity was 5.15 mg/ml and the EC 0.5 for the ferric-reducing power was >100 mg/ml (Bensmira & Jiang, 2015).
Previous studies reported that goat-milk Kefir was a promising scavenger for DPPH• radical (Baniasadi et al., 2021) and its antioxidant capacity depended on the fermentation process and storage time (Yilmaz et al., 2016).
The protective effect of Kefir on DNA damage was also studied. Figure 2 shows the electrophoretic pattern of pUT57 plasmid and could contribute to various aging diseases (Mechri, Sellem, Bouacem, Jabeur, Chamkha, et al., 2020;Mechri, Sellem, Bouacem, Jabeur, Laribi-Habchi, et al., 2020). The Kefir antioxidant potential could be explained by some LAB metabolites that showed chelating activity, reduced metal ions, and protected against oxidation reactions (Wang et al., 2017). In fact, some strains such as Lactobacillus paracasei H4-11, Lactobacillus fermentum D1-1, Lactobacillus casei H1-8, and Lactobacillus reuteri H2-12 showed high antioxidant activity including DPPH• scavenging activity and ferric-reducing ability (Liu et al., 2020). Additionally, the Kefir antioxidant activities was partly related to the proteolysis of milk proteins by Kefir microflora (Baniasadi et al., 2021). In fact, free amino acids, peptones, and oligopeptides formed following the proteolytic activity of the microorganisms of Kefir contributed to its antioxidant potential (Tagliazucchi et al., 2019). Therefore, Kefir could be used as a natural antioxidant to mitigate the reactive oxygen species production and possibly to prevent tissue damage associated with the oxidative stress.   (Hamdan et al., 2016;Makled et al., 2017Makled et al., , 2019Makled et al., 2020). Furthermore, it was reported that supplementation with Saccharomyces cerevisiae for 8 weeks improved the growth performance of Nile tilapia, which was explained by the availability of nutrients due to the immuno-stimulating compounds in the yeast (Abdel-Tawwab et al., 2020). An additional study also reported that yeast supplementation for 9 weeks could improve feed digestibility via stimulating the digestive enzymes production (Lara-Flores et al., 2003). However, a reduction in the growth of Nile tilapia exposed to 300 μg AFB1 kg −1 for 12 weeks was reported (Hassaan et al., 2020). Deng et al. (2010) showed no effect on the growth of O. niloticus and O. aureus fed an AFB1-contaminated diet (245-1641 µg AFB1 kg −1 ) during the first 10 weeks. These authors showed that the administration of this diet for 20 weeks led to a decrease in growth and HSI, as well as abnormal liver morphology (Deng et al., 2010). It seems from the obtained results and those of the literature that the reduction in tilapia body weight depends on the AFB1 dose as well as the exposure period.

| Antioxidant enzymes and lipid peroxidation
The AFB1 was known to be a potent hepatocarcinogen and hepato-      (Jia et al., 2014). The MDA level was significantly higher (p < .05) in fish fed on AFB1contaminated diet (D3), which was in accordance with previous studies on Nile tilapia exposed to AFB1. In fact, a relatively higher MDA level was measured in Nile tilapia after exposure to 300 μg/kg AFB1 for 12 weeks (Hassaan et al., 2020) or 200 μg/kg AFB1 for 3 weeks (de Freitas Souza et al., 2020). Interestingly, the Kefir administration to the fish fed on AFB1-contaminated diet for 4 weeks significantly were well known by their antioxidant potential. In fact, LAB, such as Lactobacillus fermentum, were able to reduce redox damage by a manganese SOD (Songisepp et al., 2004). Additionally, lactobacilli and bifidobacteria Kefir strains could attenuate oxidative status via the scavenged free radicals and the decrease of lipid peroxidation (Savini et al., 2010).
On the other hand, vitamins especially vitamins E and C and βcarotene, as well as exopolysaccharides, mainly Kefiran, produced during milk fermentation showed synergic antioxidant effects (Yilmaz et al., 2018). Furthermore, the reduction of oxidative stress biomarkers could be attributed to the nonavailability of a part of mycotoxins in the fish organisms. Indeed, it was reported that Kefir was able to adsorb AFB1 and consequently reduce their toxicity (Ben Taheur et al., 2017). Previous studies reported a significant correlation between pH reduction and AFB1 breakdown, since the production of organic acids and other fermentation by-products can detoxify AFB1 (Moradi et al., 2022).

| Principal component analysis (PCA)
The PCA done on measured biomarkers identified two main factors, which explained 97.09% of the total variance ( Figure 4). Factor 1 explains 76.23% of the total variance, while factor 2 explained 20.86% of the total variance confirming that D3 group fed on AFB1contaminated diet was the most affected. The AFB1+Kefir group (D4) was the least affected as compared to the AFB1 group (D3), which was in accordance with the protective effect of Kefir on the antioxidant status.
The PCA showed a negative correlation between MDA level, and SOD and GPX activities. Pearson's correlation coefficients between the studied biomarkers are presented in Table 4. A correlation coefficient higher than 0.5 was considered as significant at p < .05. In the present study, MDA level was negatively correlated with CAT activity (r = −.573), GPX activity (r = −.836), and SOD activity (r = −.677).
However, CAT, GPX, and SOD activities were positively correlated.

| Histological analysis
Post-mortem examination of all studied fish following AFB1 or Kefir showed a normal liver structure characterized by normal hepatocytes arranged as cords (Figure 6a,c). Moreover, the liver showed straight sinusoidal veins bounded by hepatocytes, as well as singular micro-lipid vacuoles. Nevertheless, histology of the liver from tilapia exposed to AFB1-contaminated diet showed marked changes, such as dilatation, disarrangement, and congestion of the sinusoids, invasion by micro-and macro-lipid vacuoles (lipid accumulation), and impaired contact between the hepatocytes and pancreatic cells (Figure 6b). These histopathological findings were similar to those reported by Abdelhiee et al. (2021). Interestingly, Kefir supplementation in the basal diet improved hepatocytes aspect, and reduced liver damage, lipid vacuoles accumulation, and sinusoid veins disarrangement (Figure 6d).

| CON CLUS IONS
The present study focused on the potentialities of Kefir supplementation in Nile tilapia exposed to aflatoxin B1 contamination, which induced liver damage. Kefir could be suggested as a probiotic food ingredient with interesting antioxidant activities capable of mitigating the harmful effect of AFB1 on Nile tilapia. It is interesting to explore the in silico and molecular exploration studies of Kefir to gain insight into the mechanisms by which the consortium carried out.

ACK N OWLED G M ENTS
Financial assistance for this study was provided by the Ministry of Higher Education and Scientific Research, Tunisia. Our thanks are due to Dr Houcine Dab (Professor in Animal Physiology at ISBAM) for his fruitful discussion.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All authors confirm that the data supporting the findings of this study are available within the article.